Commercial immunoglobulin preparations contain principally immunoglobulin G (IgG) molecules and are widely used for replacement therapy in agammaglobulinemia and hypogammaglobulinemia in passive immunization against pathogenic organisms, in prevention of Rh sensitization and in treatment of autoimmune conditions such as idiopathic thrombocytopenic purpura (ITP). The principal route of administration is by intravenous or intramuscular injection. However, high doses of immunoglobulins given intravenously have been reported to transmit NonA NonB (NANB) Hepatitis and possibly other viral infections.
Immunoglobulins are normally isolated from plasma or from placental blood by the Cohn cold alcohol procedures (see U.S. Pat. No. 2,390,074), by ion-exchange chromatography or by a variety of other published methods, which depend on the physical, chemical or immunological properties of the proteins involved.
Several procedures are being used to inactivate viruses inadvertently contaminating Factor VIII concentrates, another protein preparation made from the same human plasma pools as immumoglobulin solutions. Use of "steam", "dry heat" or "chemical" treatments, such as with beta-propionolactone (BPL) and ultraviolet irradiation or cholate/trinitro butyl phosphate (TNBP) have been attempted. A Factor VIII concentrate heated at 60.degree. C. for 72 hours in the lyophilized state (or dry heat treatment) transmitted NANB hepatitis in 11 of 13 recipients with hemophilia (see Colombo M. et al, Lancet 1985; 2:1-4). No cases of NANB hepatitis, but three cases of hepatitis B, occurred among twenty patients given a concentrate heated in the moistened state under partial steam pressure (see Schimpf K. et al, New England J. of Medicine, 316, 918 (1987)). These procedures, however, did not result in a product freed from hepatitis contamination. The use of BPL/uv irradiation, while eliminating the viral contaminant(s), may result in the chemical modification of the protein molecules and to the formation of neo-antigens which may cause clinical reactions.
Laboratory studies have shown that cholate/TNBP has been very effective in eliminating lipid enveloped viruses from coagulation products. A limited clinical study involving seven patients who had no previous exposure to blood products indicated that no transmission of hepatitis had occurred (see Horowitz, M. S. et al. Thrombosis and Hemostasis, 58 (1) p. 371 [1987]). However, the method is applicable only to the destruction of lipid-enveloped viruses. The subsequent removal of the chemicals used in this method is unfortunately difficult.
Pasteurization, that is the application of heat to solutions, is well known to be effective for the inactivation of viruses and other organisms. The application of pasteurization to milk is commonly done at 71.7.degree. C. for 15 seconds (see Pelczar M. J. et al, Microbiology, 4th Ed., McGraw-Hill, 1977, pp. 833 to 835). This treatment has been shown to effectively inactivate Mycobacterium tuberculosis, the most resistant pathogen likely present. Pasteurized normal serum albumin is not known to have transmitted any case of serum hepatitis or any other viral agent over a period of more than 35 years (see Edsall, J. T., Vox Sang.46:338-340 [1984]). The pasteurization of Factor VIII concentrate, in the presence of stabilizers such as sugars and amino acids, has greatly reduced the transmission of hepatitis (see Schimpf, K. et al, above). Pasteurization of Antithrombin III, another plasma product, in the presence of 0.5M citrate has been a required procedure for a number of years and no transmission of hepatitis has been reported following its administration.
Pasteurization of immunoglobulin solutions thus would appear to be an attractive possibility for virus inactivation. However, IgG molecules are known to aggregate upon heating in solution (see Rosenqvist, E. et al, Molecular Immunology, 24, No. 5, pp. 495-501, [1987]). In fact, heating of IgG solution at 63.degree. C. for 15 minutes is a widely used method to produce soluble IgG aggregates. Such aggregates possess properties analogous to those of antigen-antibody complexes, namely they fix complement, bind to macrophages, and induce Arthus reactions (see Christian, C. L., J. Immunology 84, 112-121). Activation of complement system by the Fc regions of aggregated IgG molecules led to many attempts to produce a form of IgG suitable for intravenous use, either by modifying the isolation procedure of IgG from plasma, or by modification or removal of the Fc region (see McClelland D. B. L., Yap P. L., Clinics in Haematology- 13, No. 1 February 1984). Hence, the general idea that immunoglobulins cannot be heated in the solution state without the formation of aggregates has been part of the immunoglobulin chemistry for many years. Fernandes, P. M. et al. (U.S. Pat. No. 4,440,679) and Hirao, Y. et al (published European Patent Application No. 0196761) claimed that immunoglobulin can be heated in solution and retain their biochemical properties, but this procedure required the presence of large quantities of sugars or sugar alcohols as a primary stabilizer (up to 50% (w/v) or higher). An additional stabilizer, which may be a neutral amino acid, a neutral inorganic salt, an organic carboxylic acid salt or a surface active agent, is claimed by Hirao et al in the above-mentioned European patent application to provide additional stability. However, the presence of large quantities of sugars as stabilizers also can stabilize viruses. Sucrose has been used in the stabilizer formulation for the preservation of rickettsiae and viruses following lyophilization (see Bovarnick et al, J. Bacteriology 59, 509-522, [1950]). Ng P. K. et al., (see Thrombosis Research 39, 439-447 [1985]) showed that the addition of sucrose offered protection against inactivation of porcine parvovirus in the pasteurization of Factor VIII solution. In addition, the removal of large quantities of sugars or sugar alcohols from the protein solution is different.